The present invention relates to a novel compound and method for preparation of polyethylene glycol (PEG) adducts of biomolecules, and particularly to PEG adducts of proteins and peptides.
Attachment of large macromolecules such as polyethylene glycol (PEG) to biomolecules such as proteins or peptides via chemical attachment is desired for modification of the properties of the proteins or peptides. Linking to PEG is referred to in the art as “pegylation”. Biomolecules circulating in the blood outside of a cell are subject to clearance, and can move through blood vessel walls (extravascularization). Attachment of relatively small biomolecules to large macromolecules can reduce extravascularization, and can enhance the in-vivo circulation half-life of the biomolecule.
Increasing half-life of the biomolecule in circulation is particularly important when the biomolecule is intended for therapeutic use. Pegylation of certain biomolecules reduces kidney clearance and spurious enzymatic degradation and immune system recognition. In “Artificial Blood”, Science, 295:1002, 1004–1005 (Feb. 8, 2002), Jerry E. Squires cites literature reports that conjugation of hemoglobin to macromolecules such as dextran, polyethylene glycol or polyoxyethylene retards the rate at which cell-free hemoglobin is cleared from blood circulation, extending intravascular dwell time up to 48 hours. The alteration of the effective solution volume of the hemoglobin through linkage to a macromolecule alters the colligative properties of cell-free hemoglobin, including osmotic pressure that appears to have a significant effect on blood pressure.
Polyethylene glycol is approved by the U.S. Food and Drug Administration for internal and topical use due to its low toxicity. Additional utilities and features of PEG-biomolecule conjugates are described in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, ed. Plenum, N.Y., 1992 and Polyethylene Glycol Chemistry and Biological Applications, J. M. Harris and S. Zalipsky, eds., ACS, Washington, 1997.
A requisite for preparation of polyethylene glycol conjugates of biomolecules is a suitably activated PEG molecule that under proper conditions reacts with a target biomolecule in an efficient, predictable manner such that the native activity of the target biomolecule is not adversely affected to a significant degree. For sensitive target biomolecules such as proteins and peptides, reactions to form PEG conjugates are best conducted in aqueous, buffered systems in order to avoid denaturation and concomitant loss of biological activity.
Most, if not all, activated polyethylene glycol compounds described in the patent and scientific literature and intended for conjugation to biomolecules such as proteins react with water in addition to the target biomolecule. See, for example, Scheme I below. Typically, the conundrum of derivatizing a target biomolecule in an aqueous medium with a water-sensitive, activated, polyethylene glycol reagent is partially solved by employing a large excess (5–10 fold) of reagent, while maintaining rigorous control of pH and temperature. An objective of any pegylation procedure is to produce pegylated biomolecules with stable, enhanced physiological properties in a predictable and reproducible manner on a meaningful, economical scale.
Scheme 1, below represents a reaction between an activated pegylation reagent of the art and a biomolecule reactant having acidic hydrogens (hydrido groups). The scheme shows the formation of hydrolysis product, pegylated biomolecule and protonated leaving group (HA). As illustrated in Scheme 1, activated pegylation reagents described in the current art often involve production of a leaving group (HA in Scheme 1 below) in addition to any hydrolyzed PEG reagent (shown as PEG-OH). The production of a leaving group presents an additional workup and purification problem during the isolation of the PEG-modified target molecule. The extent of the isolation/purification problem is influenced by the magnitude of the excess reagent employed in order to achieve the desired level of PEG modification which, in turn, is a function of hydrolysis rate of the reagent in water and the coupling rate onto a residue of the target molecule (typically —NH2 or —SH).

Typically, activated polyethylene glycol reagents described and used in the current art are acylating reagents directed toward primary amine residues (—NH2) in the target biomolecule. A common type of PEG acylating reagent is the so-called “active ester” derivative of PEG. Active ester PEGs of the N-hydroxysuccinimide (NHS), hydroxybenztriazole (HBT), imidazole (IM)and p-nitrophenol (PNP) have been described and are commercially available. (See Shearwater Corporation, Catalog 2001, Huntsville, Ala. 35801, www.shearwatercorp.com).
Reagents of the typemPEG-R—C(O)—OX,
where R=(O)C—(CH2)y or (CH2)y or (—O—),
y=zero through 4, and
X=NHS, HBT, IM or PNP
exhibit half-lives of hydrolysis of 1 minute (or less) to approximately 24 minutes at pH=8 and 25° C. Further, as half-life goes up, reactivity goes down. Recommended excesses of PEG acylating reagent vary from equal mass to 10-fold mass relative to target molecule (Shearwater Catalog 2001 p.12). Depending on the molecular weight of the target biomolecule, this recommended mass excess can be greater than a 100–1000 molar excess.
Incorporation of macromolecules such as PEG into biomolecules by using currently-available PEG acylating reagents is a demonstrably inefficient process. Problems associated with the acylating PEG reagents of the current art are exacerbated on a large scale. Variables affecting efficiency and reproducibility of pegylation procedures based on current art acylating reagents include: half-life (t1/2) of hydrolysis, pH value, temperature, time, mixing rate, nature and toxicity of leaving group, ease of product purification from leaving groups and hydrolyzed reagent, as well as the rate and extent of reactivity of the reagent toward the target biomolecule.
The rapid hydrolysis rates of acylating PEG reagents employed by the standard art preclude practical application of multi-functional, crosslinking pegylation reagents of the following type, where A is a leaving group.
A multifunctional, activated PEG reagent as shown above is useful for establishing intramolecular cross-links within protein molecules for the purpose of mapping sub-unit geographies and for stabilization of protein configurations and activities. Rapid hydrolysis rates of standard art PEG reagents, used at large molar excess favor a preponderance of “one on hits”, where one carboxyl end of the bifunctional molecule links to the target protein but the other end hydrolyzes to a carboxylic acid instead of also linking to the protein, and forming stabilizing cross-links.
In a significant advance, workers at the Albert Einstein College of Medicine of Yeshiva University N.Y., University of Iowa and BioAffinity Systems describe a novel approach for activating PEG for attachment to biomolecules that largely circumvents problems associated with hydrolytically unstable reagents. As disclosed in Acharya et al. U.S. Pat. No. 5,585,484, U.S. Pat. No. 5,750,725, U.S. Pat. No. 6,017,943, entitled “Hemoglobin Crosslinkers”, and Belur N. Manjula, et al., J. Biol. Chem., 275(8):5527–5534 (2000), a maleimide-activated PEG reagent is employed to form stable thioether bonds with an indigenous or added sulfhydryl moiety in the biomolecule. The maleimide function reacts rapidly with —SH groups without significant hydrolysis at pH 6.5–7.0 and without the production of a leaving group as is shown in the reaction below, wherein R—SH is a sulfhydryl-containing biomolecule.

Biomolecules such as a protein having a paucity of —SH groups must first be reacted with a thiolating reagent such as 2-iminothiolane (or the like) to convert native —NH2 groups from lysine into —SH groups. A practical drawback with the maleimide reagents is that they are difficult molecules to obtain synthetically.
The chemical formula of phenyl isothiocyanate, also known as isothiocyanatobenzene or isothiocyanic acid phenyl ester is shown below.
As noted in the Merck Index, 11th Ed., Susan Budavari, et al., eds., Merck & Co., Inc. (Rahway, N.J.: 1989), p. 7275, phenyl isothiocyanate is known to be used as a derivatizing agent for primary and secondary amines. Such derivatization of primary and secondary amines has typically been used for carrying out Edman degradation and amino acid analyses by HPLC.
This art does not teach the introduction of macromolecules such as polyethylene glycol by the use of a phenylisothiocyanate-containing molecule. Nor are there available any commercial sources of polyethylene glycols or other such macromolecules activated with phenylisothiocyanate. Furthermore, the precursors for obtaining such activated macromolecules are not commercially available or known in the art, e.g. agents containing both a isothiocyanate group and an isocyanate group.
The direct linkage of an alcohol-containing polysaccharide to an amine-containing protein vie reductive amination is known for linking antigenic polysaccharides to carrier proteins for the preparation of vaccines. Aldehyde groups are prepared on either the reducing end [Poren et al. (1985) Mol. Immunol., 22:907–919] or the terminal end [Anderson et al. (1986) J. Immunol., 137:1181–1186] of an alcohol-containing oligosaccharide or relatively small polysaccharide, which are then linked to an amine group in the protein via reductive amination. U.S. Pat. No. 4,356,170 discloses such preparation of useful polysaccharides that are reduced and then oxidized to form compounds having terminal aldehyde groups that can be reductively aminated onto free amine groups of carrier proteins such as tetanus toxoid and diphtheria toxoid with or without significant cross-linking. Several of the problems associated with the attachment of biomolecules to macromolecules are overcome by use of the reagents and processes described hereinafter.